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HIV-1, methamphetamine and astrocytes at neuroinflammatory Crossroads

As a popular psychostimulant, methamphetamine (METH) use leads to long-lasting, strong euphoric effects. While METH abuse is common in the general population, between 10 and 15% of human immunodeficiency virus-1 (HIV-1) patients report having abused METH. METH exacerbates the severity and onset of HIV-1-associated neurocognitive disorders (HAND) through direct and indirect mechanisms. Repetitive METH use impedes adherence to antiretroviral drug regimens, increasing the likelihood of HIV-1 disease progression toward AIDS. METH exposure also directly affects both innate and adaptive immunity, altering lymphocyte numbers and activity, cytokine signaling, phagocytic function and infiltration through the blood brain barrier. Further, METH triggers the dopamine reward pathway and leads to impaired neuronal activity and direct toxicity. Concurrently, METH and HIV-1 alter the neuroimmune balance and induce neuroinflammation, which modulates a wide range of brain functions including neuronal signaling and activity, glial activation, viral infection, oxidative stress, and excitotoxicity. Pathologically, reactive gliosis is a hallmark of both HIV-1- and METH-associated neuroinflammation. Significant commonality exists in the neurotoxic mechanisms for both METH and HAND; however, the pathways dysregulated in astroglia during METH exposure are less clear. Thus, this review highlights alterations in astrocyte intracellular signaling pathways, gene expression and function during METH and HIV-1 comorbidity, with special emphasis on HAND-associated neuroinflammation. Importantly, this review carefully evaluates interventions targeting astrocytes in HAND and METH as potential novel therapeutic approaches. This comprehensive overview indicates, without a doubt, that during HIV-1 infection and METH abuse, a complex dialog between all neural cells is orchestrated through astrocyte regulated neuroinflammation.

Introduction

Burden of HIV-1 and HAND

Worldwide an estimated 33 million people are infected with human immunodeficiency virus (HIV) and without effective treatment, HIV results in a progressive failure of the immune system. Approximately 1.1 million Americans are currently living with HIV or acquired immune deficiency syndrome (AIDS), with an estimated 50,000 new infections occurring in the U.S each year1. While the age at which neurocognitive issues develop is increasing with antiretroviral therapy (ART), ~69% of HIV+ patients continue to develop HIV-1-associated neurocognitive disorders (HAND; Matinella et al., 2015). Although the prevalence of HIV-1-associated dementia (HAD) has decreased from ~20% to less than 5% with wide-spread use of ART, other neuropsychiatric complications of HIV, including delirium, neurobehavioral impairments (depression), asymptomatic neurocognitive impairment (ANI) and minor neurocognitive disorder (MND) remain prevalent (McArthur et al., 2005; Robertson et al., 2007; Matinella et al., 2015). Significant glial activation can be found in brain tissues even in the absence of HIV encephalitis (HIVE) or even active viral replication, implicating inflammation as a causative mechanism of HAND (Tavazzi et al., 2014).

Burden of METH Abuse

Abuse of the potent psychomotor stimulant methamphetamine (METH) remains a significant public health concern as it results in neurotoxic outcomes including deficits in memory, executive function, anxiety, depression, psychosis and other neurologic manifestations (Cadet and Krasnova, 2009; Nagai and Yamada, 2010; Rusyniak, 2013). Despite declining use since 1999, 1.2 million people reported METH use in 2012, 133,000 of which were new users aged 12 and older2. An urban men's health study of over 2000 men who have sex with men (MSM) indicates that use of METH and other stimulants by MSM is 10 times greater than the general population and METH abusers are 20% more likely to contract sexually transmitted diseases, including HIV-1 (Stall et al., 2001; Rosenthal, 2006)3. METH administration occurs by various routes including oral, snorting, smoking and intravenous injection. The associated euphoria due to neurotransmitter release disappears before drug concentrations in the blood fall significantly, leading to “binging and crash” patterns of abuse, tolerance and increased METH intake2. Chronic METH exposure leads to a variety of adverse physiological consequences including skin lesions, tooth decay, weight loss, altered immunity, and cognitive impairment. It has been estimated that 40% of METH users exhibit global neuropsychological impairment (Rippeth et al., 2004).

METH and HIV-1 Comorbidity

Eight percent of newly diagnosed HIV-1 infections are attributed to intravenous drug use and the National Institute on Drug Abuse reports that 25% of diagnosed HIV-1-infected individuals report treatment for the use of drugs and alcohol4. While accurate statistics documenting METH abuse in HIV-1-infected individuals are not available, studies show that METH use exacerbates HIV-1 infection, accelerating the severity and onset of HAND, along with immune dysfunction and resistance to ART therapy (reviewed in Passaro et al., 2015) Studies report that 53–58% of HIV+ METH users exhibit neurocognitive impairment compared to 40% in either HIV+ or METH+ alone; however, their interaction is poorly understood (Rippeth et al., 2004; Gupta et al., 2011). In part, the neurological complications in both METH abuse and HAND are associated with increased permeability of the blood brain barrier (BBB) and neuroinflammation. These are mediated through cellular and molecular mechanisms such as gliosis, viral replication, oxidative stress and excitotoxicity (Rippeth et al., 2004; Ramirez et al., 2009; Potula et al., 2010; Sharma et al., 2011; Cisneros and Ghorpade, 2012).

The study of inflammation generally focuses on the contributions of professional immune cells. However, the unique nature of the brain as an immune privileged site implicates neural cells in the regulation of immune responses. Glia, specifically astroglia and microglia, account for 50–80% of the cells in the brain, outnumbering neurons in certain regions by as much as 10:1 (Dobbing and Sands, 1973; Kandel et al., 2000; Azevedo et al., 2009). As the resident immune cells of the brain, microglia are accountable for brain surveillance and immunity, while astrocytes have a significant repertoire of immune functions that complement their “neural” functions. Astrocytes, through regulation of neuroinflammation, are implicated in neurodegenerative diseases such as Alzheimer's disease (AD; Roth et al., 2005; Weinstein et al., 2013), hepatic encephalopathy (Coltart et al., 2013), multiple sclerosis (MS; Brosnan and Raine, 2013; Kostic et al., 2013), epilepsy (Devinsky et al., 2013), amyotrophic lateral sclerosis (Evans et al., 2013), Parkinson's disease (PD; Tufekci et al., 2012), aging and depression (Paradise et al., 2012) and HAND (Borjabad et al., 2010; Cisneros and Ghorpade, 2012). Reactive glia participate in neuroinflammation by synthesizing and releasing various powerful pro-inflammatory and neuroactive substances, like cytokines, chemokines, nitric oxide (NO), glutamate, reactive oxygen species (ROS), neurotrophins and transforming growth factors (TGF; da Cunha and Vitkovic, 1992; Chiueh and Rauhala, 1999; Wang et al., 2004; Hult et al., 2008; Fitting et al., 2012; Ramesh et al., 2013; Salamanca et al., 2014). Although astroglia play a central role in HIV-1-associated neuropathogenesis, serving as reservoirs for latent HIV infection, chronic inflammation and as sources of neurotoxicity during HAND. There is a paucity of information regarding the mechanisms at play during HIV-1 and METH comorbidity. Due to the addictive nature of METH abuse, METH interactions with neurons leading to dopamine release and subsequent toxicity have been a focus of much investigation. However, despite apparent glial activation, the mechanisms through which METH interacts with glia to alter astrocyte and microglial function are much less apparent (Chiu and Schenk, 2012; Cisneros and Ghorpade, 2012; Friend and Keefe, 2013). A better understanding of astrocyte regulation of HIV-1 and METH-mediated neurodegeneration would help identify mechanistic targets coordinating glial activation. By therapeutically reducing acute and chronic inflammation, neurological impairments such as HAND could be ameliorated or even prevented.

Astrocytes in HAND

As a predominant cell in the brain, astrocytes regulate the central nervous system (CNS) physiological environment at both the BBB and in the parenchyma. As integral members of the BBB, astroglia respond to immunomodulatory signals including, but not limited to, cytokines and prostaglandins. During HIV-1 CNS infection, the BBB integrity is compromised thus permitting the peripheral immune system to trigger neuroinflammation and oxidative stress. Astrocytes secrete a variety of neuroactive molecules in response to HIV-1- and METH-associated stimuli. In this manner, astrocytes regulate the physiological functions of neural cells in their immediate vicinity and cells within the reach of their many foot processes. As neuroinflammation persists, the ability of astrocytes to regulate BBB integrity, and neurotransmission in tripartite synapses is impaired. Under chronic disease, astrocyte expression of critical transporters and enzymes that clear neurotransmitters, neutralize ROS and balance ECM remodeling dwindles to levels where homeostasis is no longer sustainable. Eventually, neuronal function and survival are impaired due to insufficient support and direct toxicity. Taken together, astrocyte dysfunction during METH abuse, in the setting of HIV-1 infection, contributes both to chronic BBB damage and propagation of a CNS environment dominated by inflammation, oxidative stress, and excitotoxic insults, that culminate in neurodegeneration.

The entry of HIV-1-infected cells into the brain is the foundation of HIV-1-associated neurodegeneration; however, the outcome of HIV-1 CNS infection varies dramatically between individuals. Even before ART, disease progression to AIDS with and without neurocognitive impairment could take years. However, METH abuse exacerbates HIV-1-associated disease pathology, inducing changes that may last for decades even after METH is no longer abused (Cadet and Krasnova, 2007; Iudicello et al., 2014; Northrop and Yamamoto, 2015). HIVE, the most severe form of HAND, is pathologically characterized by inflammatory changes and accumulation of perivascular MP, formation of microglial nodules and multinucleated giant cells, astrogliosis, neuronal atrophy and death (Gendelman, 2005). With the effective use of ART helping to suppress disease progression, clinicians and researchers alike postulate that ANI and MND are stages of a similar disease process (Strazza et al., 2011). However, since HAND is often a comorbidity rather than the cause of death, HIV-1-associated neuropathology is often “non-specific,” leading many to search for other more subtle mechanisms of neurodegeneration (Gelman, 2015). Neuroinflammation remains a focus of intense study as inhibiting viral replication alone has slowed, but not halted, HAND progression.

Neuroinflammation

The pro-inflammatory cascade leading to the disruption of the BBB and entry of HIV-1-infected leukocytes into CNS continues in the brain microenvironment. Resident microglia and perivascular MP perpetuate neuroinflammation, activating and or transmitting the infection to non-infected cells, including astroglia. As the resident immune cells, microglia are the primary HIV-infected cells in the brain mediating neuroinflammatory responses, by increasing cytokines, MMPs and cytotoxic factors (Ramesh et al., 2013). However, microglial activation and infection inevitably also lead to astrocyte activation and infection of a very small percentage of astrocytes with HIV. HIV infection in astrocytes is restricted to the extent that are capable of expressing viral proteins, including gp120, Tat and Nef, but not infectious virions (Messam and Major, 2000; Eugenin et al., 2011; Fitting et al., 2012; Li et al., 2015; Luo and He, 2015). Coculture experiments mimicking the interconnections between BMVEC and astroglia demonstrate that a small percentage (4.7%) of HIV-1-infected astrocytes can lead to endothelial apoptosis, dysregulation of lipoxygenase/cyclooxygenase (COX), calcium (Ca2+) channels and ATP receptor activation within astrocytes, significantly contributing to BBB disruption (Eugenin et al., 2011). Further, astrocytes exposed to HIV-1 proteins, along with those expressing them, have been shown to modulate to neuroinflammation through multiple regulatory pathways, summarized in Tables 1, 2.

Pro-inflammatory molecules also propagate inflammation by the spread of reactive gliosis and affect neuronal function and survival by direct and indirect mechanisms. In the healthy nervous system, cytokines and chemokines are neuromodulators, regulating neurodevelopment, neuroinflammation, and synaptic transmission. They are fundamental to the brain's proper immune function, serving to maintain immune surveillance, facilitate leukocyte traffic, and recruit other inflammatory factors (Chui and Dorovini-Zis, 2010). However, during neuroinflammation associated with both HIV-1 infection and METH exposure, activated glia mediate neuronal injury and death through neurotoxic signaling, generation of ROS, altered cellular metabolism, neurotransmission and cerebral blood flow, among others (Lau et al., 2000; Abdul Muneer et al., 2011; Hoefer et al., 2015). In such, reactive glia, infected or not, participate in the disruption of the BBB leading to infiltration of HIV-1-infected cells into the CNS and continuation of neuroinflammation in the brain. The specific contributions and regulation of these cytokines, chemokines and bioactive molecules in reactive astrocytes and other cells during HIV-1 and METH comorbidity are summarized in Tables 1, 2 and will be discussed in more detail below.

Gliosis

Although infiltration of peripheral immune cells often leads to significant neural damage, leukocyte infiltration is not always associated with neurotoxicity (Boztug et al., 2002; Trifilo and Lane, 2003; Clark et al., 2011). In such, the resident glial cells, microglia and astroglia, are implicated as central players in the inflammatory responses associated with neurodegeneration. The term gliosis refers to a non-specific reactive change in glial cells in response to damage, disease or infection in the CNS. Reactive glia are often identified in brain tissue by morphological changes, including increased size, elongation of processes and increased reactivity with cellular markers. Morphological changes are indicative of altered glial function. The primary goal of gliosis is to restore brain homoeostasis by providing trophic support, tissue repair and containment of the affected region. As discussed above, reactive glia secrete many neuroactive substances capable of injuring neural cells, dependent upon the type, severity and duration of insult. Ultimately, the balance between the beneficial and detrimental effects of gliosis determines the outcome in the CNS.

Microglia

Microglia make up between 10 and 15% of neural glia and are accountable for the innate immune response in the brain (Lawson et al., 1992; Verkhratskiǐ and Butt, 2013; Elmore et al., 2014). The homeostatic functions of microglia tend to go unnoticed in the brain, even though they play an active role in embryonic brain development and clear neuronal or glial debris, while surveying their environment for threat and injury (Beyer et al., 2000; Casano and Peri, 2015). When injury or infection is detected, microglia undergo dramatic morphologic alterations, shifting from resting ramified cell into an activated amoeboid phenotype, and transforming into a more classically functioning immune cell (Burdo et al., 2013; Tavazzi et al., 2014). Activated microglia upregulate surface receptors, including major histocompatibility complex molecules, leading to secretion of factors that influence neuronal survival and a chronic neuroinflammatory state (Streit, 2000; Block and Hong, 2005). Reactive microgliosis is associated with the pathogenesis of many common types of neurodegeneration, including HAND (da Fonseca et al., 2014; Pasqualetti et al., 2015).

METH Abuse: Implications for Astrocytes as Viral Reservoirs

HIV-1 can invade the CNS early during infection, primarily infecting infiltrating monocytes and resident microglia, along with a small proportion of astroglia. HIV-1 then integrates with the host cell genome as a provirus, leading to both active and latent infection. During active HIV-1 infection in permissive cells, budding of infectious virions ensues. However, in non-permissive cells such as astrocytes, active HIV-1 infection is restricted to expression of viral proteins, which are incapable of maturing into infectious particles (Messam and Major, 2000; Eugenin et al., 2011; Fitting et al., 2012; Li et al., 2015; Luo and He, 2015). Viral replication is limited in astrocytes at various steps of the virus life cycle including virus entry, reverse transcription, transport and translation of viral RNA, and maturation of progeny virions (reviewed in Messam and Major, 2000; Gorry et al., 2003). Other studies suggest that if restrictions on viral entry into astrocytes are bypassed, the intracellular environment may be conducive to productive viral infection (Canki et al., 2001; Chauhan, 2014).

In the brains of HIV-1-infected individuals with METH dependence, epigenetic changes were associated with increased global DNA methylation as compared to the brains of HIV-1+ individuals without METH use. METH exposure led to differential methylation in genes connected to neurodegeneration, oxidative phosphorylation, dopamine metabolism and transport (Desplats et al., 2014). Differential regulation of gene expression in microglia and astrocytes during METH and HIV comorbidity may induce viral replication and expression of pro-inflammatory mediators to contribute to neurodegeneration. METH enhances viral replication in macrophages and may upregulate or downregulate infection in T cells (Liang et al., 2008; Wang et al., 2012; Mantri et al., 2014). METH activates transcription of proviral DNA in latently HIV-1-infected human microglial cells, leading to activation of the NF-κB signaling pathway (Wires et al., 2012). Feline immunodeficiency virus (FIV), a lentivirus related to HIV-1, leads to astrogliosis and microgliosis. METH has been shown to increase cell-associated FIV replication in feline astrocytes and cell lines (Phillips et al., 2000; Gavrilin et al., 2002). Reactivation of viral expression in latently infected astrocytes could contribute to either increased neuroinflammation and toxicity or the elimination of viral reservoirs by viral cytopathic effects and lysis by effector cells. During METH, adherence to ART is decreased and the immune system is depressed (Reback et al., 2003; In et al., 2005), tipping the balance toward increased HIV-1- and METH-associated neurodegeneration. A quick, wide-spread activation of latently infected cells, coupled with effective ART delivery to counter the spread of infection, may lead to the clearance of HIV-1-infected neural cells (Díaz et al., 2015). However, the implications of widespread elimination of infected astrocytes and other latently infected cells on neural function are unknown; the results of which may favor strategies for maintaining a latent CNS infection, rather than radical activation and elimination. (reviewed by Brew et al., 2013; Churchill and Nath, 2013).

Astrocyte Interactions with HIV-1 Virions, Proteins, and METH

In astrocytes, expression of and exposure to virus, HIV-1 proteins, such as gp120, Tat, Nef, or Vpr, and HIV-1-relevant cytokines induce a host of factors that influence neuronal survival and function (Table 2). Both HIV-1 and METH alter astrocyte expression of inflammatory mediators, neurotransmitter receptors and transporters, which in turn alter the brain microenvironment, leading directly and indirectly to neuronal dysfunction or death. HIV-1-relevant cytokines also regulate astrocyte cytotoxicity, function and glia-neuron crosstalk during HIV-1 infection and METH abuse.

Behavioral testing in transgenic mice expressing HIV-1 gp120, under the control of the GFAP promoter, with and without METH administration, showed impaired learning and memory and increased disinhibition even after months of METH abstinence (Hoefer et al., 2015). Both METH and gp120 alone lead to loss of dendrites and presynaptic terminals, along with reduced long-term potentiation, which is associated with learning and memory. Further, post-tetanic potentiation, a measure of synaptic plasticity, was also decreased in METH-treated, gp120-transgenic mice (Hoefer et al., 2015).

Common Signaling Pathways

A large majority of bioactive molecules discussed above facilitate communication among various CNS cells.

Signals received by target receptors regulate astrocyte function during HIV-1 and METH-associated neuroinflammation through a variety of cross-linking pathways. As IL-1β is a prototypical cytokine for astrocyte activation, the NF-κB pathway contributes to the regulation of many astrocyte genes and is involved in cellular responses to stimuli such as stress, cytokines, free radicals, glutamate or viral antigens (reviewed in Mémet, 2006). Downstream of the IL-1 receptor (IL-1R), the IκB kinase complex phosphorylates and degrades the NF-κB sequestering protein, IκBα, leading to NF-κB release. Persistent NF-κB activation is implicated in the prolonged induction of selective pro-inflammatory genes in human glial cells (Griffin and Moynagh, 2006). The mitogen activated protein family of kinases (MAPK), including extracellular signal-regulated kinases (ERK), c-Jun N-terminal kinases (JNK) and p38, also regulate many HIV-1- and METH-induced astrocyte responses, which often culminate in NF-κB-mediated gene transcription (Table 2). IL-1β signaling can also be negatively regulated by expression of inhibitory type IL-1R, IL-1R antagonist, soluble and decoy receptors. Dysregulation of the IL-1β system in the brain has been implicated in AD, MS and epilepsy (Garlind et al., 1999; Ravizza et al., 2006; Dujmovic et al., 2009) Cytokine receptors for IFNs and a few ILs are coupled to the JAK/STAT pathway. Here, JAK phosphorylation of various tyrosine kinases facilitates STAT dimerization and gene transcription. METH- and Tat-induced astrogliosis and GFAP expression are also regulated through STAT3 (Robson et al., 2014; Fan et al., 2015) Ligation of G-coupled receptors such as CXCR4 can differentially initiate downstream elements including cAMP and [Ca2+]i to mediate function. CXCL12 and gp120 induce ERK 1/2 activation in human neurons, while only CXCL12 did so in astrocytes (Griffin and Moynagh, 2006). Induction of differential signaling pathways also influences HIV-1 gene transcription in astrocytes, where TGF-β-linked transcription factors, Smad3 and 4, interact with C/EBP-β to offset Tat-mediated LTR activity (Coyle-Rink et al., 2002).

A consequence of extended activation of neuroinflammatory signaling cascades is Ca2+ dysregulation in both glia and neurons. Intracellular Ca2+, when released from the ER, acts as a secondary messenger and regulates the activity of many enzymes, ion channels and cytoskeletal components. In astrocytes, [Ca2+]i signaling is induced by activity in adjacent neurons, glutamate, ATP, METH and HIV (Banerjee et al., 2008; Reddy et al., 2012). Dysregulation of [Ca2+]i is implicated in astrocyte Aβ-associated neurotoxicity and ischemia, through Ca2+-mediated glutathione depletions and voltage-gated Ca2+ influx (Duffy and MacVicar, 1996; Abramov et al., 2003). These various routes of Ca2+ signaling converge on a common pathway involving Ca2+ overload-induced mitochondrial dysfunction, including oxidative stress, cytochrome c release and injury or apoptosis in neurons and astrocytes alike (Stanika et al., 2009; Eugenin and Berman, 2013).

Therapeutics to Target Astroglia

The various roles of astroglia in CNS pathology are only beginning to be defined and reactive gliosis is now well recognized as a ubiquitous feature of CNS pathologies. Astrogliosis is not a simple on or off switch, but rather a finely tuned continuum of molecular, cellular and functional alterations. These changes in gene expression and function can exert both beneficial and detrimental effects in the brain milieu, dependent upon the duration and context of the specific molecular signaling cascades. Glial activation and dysfunction are emerging as important targets during neuroinflammation (Jha and Suk, 2014). Astroglia actively participate in neurodegeneration through the loss of normal functions and gain of abnormal activities. The ever-expanding understanding of the mechanisms regulating these changes has the potential to identify many molecules that may serve as therapeutic targets for neuroinflammatory disorders including METH abuse and HAND (Table 3).

TABLE 3

Table 3. Therapies targeting astroglial activation and function.

US Food and Drug Administration (FDA) Approved Medications

Medications already in use for non-HIV/METH/astrocyte related therapies have shown changes in HIV-1- or METH-associated neuroinflammation, glial activation and neurotoxicity. Tricyclic antidepressants, such as clomipramine, are cited in the 2015 WHO model list of essential medicines needed in a basic health system to treat anxiety and depressive disorders by inhibiting serotonin and norepinephrine reuptake6. However, in a recent study on microglia and astrocyte cultures both clomipramine and imipramine reduced NO, iNOS, IL-1β and TNF-α expression by inhibiting IκB degradation, NF-κB p65 translocation to the nucleus and phosphorylation of p38 MAPK (Hwang et al., 2008). When used in microglia-neuroblastoma cocultures, both antidepressants significantly reduced glia-mediated-cell death (Hwang et al., 2008).

Fingolimod, an immune modulating drug used to treat MS, decreases astroglial activation and NO production in response to sphingosine-1-phosphate (S1P), IL-1β and IL-17 (Colombo et al., 2014). Fingolimod modulates autoimmune lymphocyte release from the lymph node by agonizing the S1P receptor, and also prevents monocyte: endothelial interactions (Bolick et al., 2005; Baumruker et al., 2007). However, in astrocytes fingolimod also decreased IL-induced, NF-κB-mediated signaling and reduced neurotoxicity following transfer of conditioned supernatants from activated astrocytes. Further, in an experimental autoimmune encephalomyelitis mouse model, fingolimod hampered astrocyte activation and NO production (Colombo et al., 2014). These results indicate that fingolimod can traverse the BBB and/or decrease monocyte infiltration into the CNS, supporting it as a candidate to decrease glial activation during HAND. However, these positive effects on glia would have to be balanced with impaired lymphocyte maturation in the lymph node. Copolymer-1, another MS drug that serves as a myelin decoy, showed anti-inflammatory benefits in an HIVE mouse model, with decreased pro-inflammatory cytokine and iNOS expression, coupled with increased BDNF levels. Both microgliosis and astrogliosis were reduced with treatment, which correlated with diminished neurodegeneration (Gorantla et al., 2007, 2008). These and other glial modulating, MS drugs may warrant future therapeutic consideration for HAND.

Another class of plant metabolites, known as flavonoids, are found in tea, red wine, dark chocolate, Ginkgo biloba and berries (Haytowitz8). Research into their potential broad health benefits against oxidative stress, inflammation, cancer and cardiovascular disease is currently ongoing; yet, no health claims have been approved by the FDA or European Food Safety Authority for use as pharmaceutical drugs (Agostoni et al., 2010). However, flavonoids such as silibinin have been shown to possess anti-HIV-1 and HCV effects in T-cells by blocking viral replication, cell activation and proliferation (McClure et al., 2012). Orally administered anti-oxidants, such as flavonoids, have the capacity to inhibit microglial migration, ROS and IL-1β production, AA- and COX-2-mediated inflammation and toxicity (Nanda et al., 2007; Chuang et al., 2014; Singh and Pai, 2015). Assessment of ROS/RNS-mediated post-translational modifications of brain proteins in the CSF and brain tissues may reveal biomarkers associated with HIV-1-neurodegeneration (Uzasci et al., 2013). Biomolecules available in food by targeted dietary changes or supplementation that exert both generalized anti-oxidant and anti-inflammatory effects could penetrate the brain and reduce glial activation.

Receptor Antagonists

Astrocyte activation during METH abuse leads to persistent increase in GFAP immunoreactivity and reactive phenotypes even months after cessation of METH abuse. Therapeutic targeting of METH signaling receptors in astrocytes may reduce astroglial activation and impaired astrocyte function. In-depth studies on neuronal METH receptors have led to significant insight into the addictive and euphoric effects of METH abuse. In astrocytes; however, there is a paucity of these investigations with few recent reports that document METH receptors on astrocytes (Cisneros and Ghorpade, 2014; Robson et al., 2014; Zhang et al., 2015).

During METH exposure, trace amine associated receptor 1 (TAAR1) modulates dopamine levels in the synapse by regulating DAT activity in neurons. Activation of TAAR1 by METH stimulates protein kinase (PK)A and PKC to phosphorylate DAT. It has been proposed through studies in TAAR1 KO mice that phospho-DAT either acts in reverse, effluxing dopamine into the synapse, or is internalized, preventing dopamine reuptake from the synapse (Miller, 2011). TAAR1 is also expressed in primary human astrocytes, lymphocytes, B-cells and is upregulated during activation with METH and pro-inflammatory mediators (Panas et al., 2012; Babusyte et al., 2013; Cisneros and Ghorpade, 2014). In astrocytes, TAAR1 is upregulated during METH/HIV-1 cotreatment. Further, astrocyte TAAR1 activation by METH increases cAMP levels and downregulates EAAT-2 expression and function, which may lead to excitotoxicity and neuronal dysfunction or death due to impaired glutamate clearance from the synapse by astrocytes (Cisneros and Ghorpade, 2014). METH-induced alterations in EAAT-2 expression and function were blocked by TAAR1 knockdown, implicating TAAR1 as a therapeutic target for astrocyte-mediated neurotoxicity during METH and HIV-1 neurodegeneration (Miller, 2012; Cisneros and Ghorpade, 2014). In lymphocytes, METH-induced phosphorylation of PKA and PKC could be significantly reduced by EPPTB, a selective TAAR1 antagonist/reverse antagonist (Miller, 2012; Panas et al., 2012). However, TAAR1 KO mice show increased sensitivity to METH as measured by striatal dopamine release and augmentation of METH-induced behaviors (Wolinsky et al., 2007; Lindemann et al., 2008; Achat-Mendes et al., 2012). TAAR1 overexpression in the neurons of transgenic mice decreased sensitivity to amphetamine, even with increased extracellular dopamine levels in the accumbens nucleus and serotonin in the medial prefrontal cortex (Revel et al., 2012). Interestingly, attenuation of TAAR1 activation with a selective partial antagonist, RO5073012, restored METH-mediated changes in locomotor activity. Therefore, constitutive or tonic activation of TAAR1 by natural agonists may regulate physiological monoamine activity in neurons (Revel et al., 2012). TAAR1 agonists also suppress hyperactivity and improve cognition in glutamate receptor deficiency models (Revel et al., 2011, 2013) and TAAR1 modulates cortical glutamate NMDA receptor function in TAAR1 KO mice (Espinoza et al., 2015). Thus, a balance between agonism of neuronal TAAR1 and antagonism of astrocyte TAAR1 will need to be further investigated to balance the neuroprotective benefits of TAAR1 targeting drugs.

In this review, we have provided an in-depth summary of the existing literature about METH effects on astrocytes in the setting of HIV. This comprehensive overview indicates, without a doubt, that astrocyte regulation of neuroinflammation during HIV-1 infection and METH abuse involves a complex dialog between all neural cells. Figure 1 provides a graphic summary of ongoing events and a proposed temporal order for these activities. (1) As HIV-1 and METH gain access to the brain across the BBB, they interact with astrocytes and induce production of reactive oxygen and nitrogen species. (2.1) These along with cytokines and chemokines from either side of the BBB, act to increase BBB permeability. Chemokine gradients recruit leukocytes, which bring HIV-1 and inflammation as they extravagate into CNS. Brain microglia and perivascular macrophages, when activated and infected, secrete cytokines, virus, viral proteins and ROS, which in turn activate astrocytes to perpetuate (2.2) neuroinflammation and (2.3) oxidative stress. In response to activation, astrocyte EAAT-2 levels decrease and extra cellular glutamate levels rise. (2.4) Pathological glutamate levels overexcite neurons impairing function through excitotoxicity. (3) Concurrently, METH and neuroinflammation activate astrocytes and microglia in the vicinity, instigating reactive gliosis. (4) METH and other pro-inflammatory cytokines can activate proviral gene expression in astrocytes and microglia. (5) Infected glia secrete viral proteins and pro-inflammatory mediators, which alter astrocytes homeostatic functions and perpetuate neuroinflammation. Cytotoxic molecules, including cytokines, viral proteins and ROS, coupled with depletion of astrocytic neurotrophic support, induce neuronal dysfunction and death. (6) Intervening with therapeutics targeting astroglia may disrupt the neuroinflammatory dialogue and protect neurons during HAND and METH abuse.

Taken together, this comprehensive review further emphasizes that additional studies regarding glial-based mechanisms/interactions, implicated in the combined setting of METH and HIV, are timely and highly warranted. Moreover, this review presents a platform to persuade future investigators to examine several critical questions that remain unanswered and are likely to influence therapeutic outcomes. Perhaps, most importantly, it is yet unknown how these interactions differ in the setting of long-term ART. Are there any disparities related to the outcomes of the combined interplay outlined in Figure 1 in the setting of race and/or gender? Epigenetic factors may play a significant role in these phenomena and we have only begun to scratch the surface of the role of genetic background and/or predisposition. Over the next several years, HIV-associated comorbidities including neurological and metabolic complications and related astroglial contributions, will continue to hold high research priorities. While we have highlighted several salient features of astroglial contributions to neuroinflammation, the role of METH and other drugs of abuse in this setting will continue to unravel. Continued elucidation of the regulatory mechanisms governing astroglial responses to METH and HIV-1 will provide the foundation for the generation of novel therapeutic interventions for neuroinflammatory disorders by targeting a key player, astrocytes.

Funding

The studies were supported by grant R01DA039789 from NIDA to AG.

Conflict of Interest Statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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